Protective effect of Tribulus terrestris fruit extract on cerulein-induced acute pancreatitis in mice.

OBJECTIVE
Antioxidant, anti-inflammatory, analgesic and antimicrobial activities of Tribulus terrestris (T. terrestris) could be helpful in the treatment of acute pancreatitis; thus, this study was designed to investigate the effects of T. terrestris on cerulein-induced acute pancreatitis in mice.


MATERIALS AND METHODS
Three doses (100, 200 and 400 mg/kg) of T. terrestris hydro-alcoholic extract were administered both orally (60 minutes before pancreatitis induction, p.o.) and intra-peritoneally (30 minutes before pancreatitis induction, i.p.) to different groups of mice (n=6). Pancreatitis was induced by ﬁve injections (i.p.) of cerulein 50μg/kg body weight with 1 hr intervals. Animals were euthanized 5 hr after the last injection of cerulein and tissue injures were assessed biochemically and pathologically.


RESULTS
T. terrestris extract 200 and 400mg/kg (p.o.) and T. terrestris extract 400 mg/kg (i.p.) reduced pancreatic tissue myeloperoxidase (MPO) activity and serum amylase and lipase levels and alleviated histological parameters.


CONCLUSION
These data suggest that T. terrestris hydro-alcoholic extract was effective in protecting against experimental acute pancreatitis and possibly the efficacy depends on dose and route of administration.


Introduction
Acute pancreatitis (AP) denotes a potentially lethal disorder which is characterized by persistent inflammation of the pancreas over a short period of time as well as elevation in serum level of digestive enzymes without specific therapy (Spanier et al., 2008).
It seems that acute pancreatitis could be potentially life threatening. The morbidity and mortality associated with pancreatitis are secondary to cardiogenic shock, cardiac, renal and respiratory insufficiency and hepatic dysfunction. Also, it can lead to progression of failure in distant organs which denotes multiple organ failure (Lankisch and Banks, 2013).
Some drugs like ACE-inhibitors, statins, estrogens, diuretics, highly active antiretroviral treatment (HAART), and valproic acid have been also implicated in drug-induced acute pancreatitis with limited understanding of their mechanisms (Badalov et al., 2007;Kaurich, 2008).
The early pathophysiology of the acute pancreatitis has not been well understood, but some clinical studies have shown that after an initial acinar cell injury, proinflammatory cytokines such as interleukin (IL) 1, IL-6, IL-8 and tumor necrosis factor alpha (TNF-α) are increased in the serum of patients with acute pancreatitis (Pooran et al., 2003) while the degree of cytokine elevation correlates with disease severity (Raraty et al., 2004).
Current treatment of pancreatitis is largely supportive and consists of different combination therapy including: antibiotics, fluid resuscitation, nutritional support and pain control. Since there is no single drug or therapy known to treat this disease effectively, a great need exists to find new therapies for acute pancreatitis (Kambhampati et al., 2014).
It seems that medicinal herbs are suitable alternatives for treatment of acute pancreatitis as they have various components that can cover different aspect of pancreatitis pathophysiology as it has been shown in various studies Genovese et al., 2006;Minaiyan et al., 2014b;Qiong et al., 2005).
Tribulus terrestris (Caltorps) is belonging to Zygophillaceae family which consists of 20 species around the world. Caltorps is an annual plant adapted to grow in dry climate locations through India, China, parts of Europe and Iran (Kianbakht and Jahaniani, 2003).
T. terrestris extract is well patronized in Iranian and Indian traditional medicine texts like Ayurvadic text for different medicinal uses like anti-inflammatory, nutritive, diuretic, anti-dysuria, hepatoprotective and pain alleviating purposes. The extracts of the fruits and leaves also have cardiac stimulant and fertility potentiating activity and may improve sexual activity (Chhatre et al., 2014;Neychev and Mitev, 2005). Furthermore, antioxidant, analgesic, antiinflammatory and antibacterial properties of T. terrestris fractions have been demonstrated in several experimental investigations (Baburao et al., 2009;Heidari et al., 2007a;Mitra et al., 2012;Zheleva-Dimitrova et al., 2012).
T. terrestris exhibits a promising safety profile as the maximum tolerated dose of 50% ethanol extract of fruits in mice was 100 g/kg (Shaheen et al., 2012). Its safety in humans has also been shown; so, the T. terrestris extract as a drug supplement is found in market (Rogerson et al., 2007).
These data suggest that T. terrestris might possess protective effects against pancreatitis; so, the present study was performed to evaluate the protective effects of T. terrestris against ceruleininduced acute pancreatitis in mice to have a better insight into the mechanism (s) of actions of T. terrestris on pancreatitis.

Plant material and extraction
T. terrestris fruits were collected from the local areas of Fereydan, Isfahan province, Iran. The plant identity was confirmed by Pharmacognosy Department of School of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran. The fruits were cleaned, dried and grinded and the dried powder was extracted with ethanol: water (70:30) using maceration method. The soaked powder was kept at room temperature for 24 hr with shaking, then, it was filtered. This process was repeated three times. Subsequently, all the filtrate was pooled, evaporated to dryness in a rotary evaporator to yield a semi-solid extract. Then, this extract was freeze-dried under reduced pressure and stored in the refrigerator till use (Hajhashemi et al., 2010).

Determination of total phenols
The total phenolic content of freezedried powders of T. terrestris fruit was checked by Folin-Ciocalteau micromethod by plotting the standard curve using gallic acid solutions (50, 100, 150, 250 and 500 mg/L) as reference agent.
A total volume of 20 μl of blank, standard and sample solutions were separately added into tubes and to each tube, 1.58 mL of distilled water was added. Then, 100 μL of Folin-Ciocalteu Reagent (Sigma, St. Louis, MO, USA) was added and mixed well. After 8 min, 300 μL of sodium carbonate solution 20% was added and mixed. The solutions were maintained at 40°C for 30 min and the absorbance of solutions was detected at 760 nm against the blank using ultra violet (UV)-Vis spectrophotometer (Waterman and Mole, 1994).

Animals
Sixty male mice (12 weeks old, 30-40 g) were used. Mice were housed in plastic mouse cages (6 in each) with controlled temperature, humidity, and light/dark cycles (12hr/12hr) and had free access to pelleted rodent chow and tap water.
Before initiation of the experiment, the animals were fasted over the night for 12 hr. The study was authenticated by the Ethics survey for Animal Care and Uses, Isfahan University of Medical Sciences, Isfahan, Iran.

Grouping
Animals were randomly divided into the following 10 groups (n=6).
Sham group: Twelve mice were treated p.o. and/or i.p. with 10ml/kg body weight (B.W.) normal saline without any pancreatitis induction.
Control groups: Twelve mice were treated p.o. and/or i.p. with 10ml/kg B.W. normal saline 60 and 30 min before pancreatitis induction, respectively.
Parenteral extracts treated groups: Eighteen mice were treated with 100, 200 and 400 mg/kg B.W. (i.p.) of hydroalcoholic extract of T. terrestris 30 min before pancreatitis induction. Then, 5 hr after the last injection of cerulein, blood samples were taken by intra-neck puncture after head blowing and stored at -20 C for biochemical analysis till 2 weeks.
The pancreas was quickly removed and fixed in formaldehyde (10%) for histological examination. Besides, portions of this organ were immediately frozen in liquid nitrogen and stored at -70 • C until assayed for myeloperoxidase (MPO) activity. Microscopic image was captured by a pathologist unaware about the study protocol using a professional camera (Sony®, Japan) set on an optical microscope (Minaiyan et al., 2014b).

Amylase and lipase activity analysis
Serum lipase and amylase activity were calculated using commercially available lipase and amylase kits (Pars-Azmoon Company, Tehran, Iran) (Minaiyan et al., 2014b).

Myeloperoxidase activity assay
MPO activity, an index of polymorphonuclear leukocyte accumulation and oxidative stress, was determined according to the modified method reported by Bradley et al. (1982).
Pancreas tissue was homogenized in 1mL of 50 mM potassium phosphate buffer containing 0.5% HTAB (hexadecyl trimethyl ammonium bromide). Then, the homogenate was homogenized in an ice bath for 10 s, freeze-thawed thrice with sonication between cycles.
The suspensions were centrifuged at 15,000 rpm for 15 min at 4 • C and then, the supernatant (0.1mL) was allowed to react with 2.9 mL of 50 mM potassium phosphate buffer (pH 6.0) containing Odianisidine dihydrochloride (0.167mg/mL) and 0.005% hydrogen peroxide.
The absorbance of the reaction mixture was measured at 450nm using a UV-Vis spectrophotometer. MPO activity was expressed in units (U) per gram of wet tissue weight (Minaiyan et al., 2014a).

Statistical Analysis
In this study, all the statistical analyses were performed by GraphPad Prism ver 5.04. Biochemical quantitative data are expressed as mean±SEM. Graded data are expressed as median (range) values.
Statistical analysis was carried out using one-way analysis of variance (ANOVA) followed by Tukey's multiple comparison tests. Nonparametric data was analyzed by Mann-Whitney U test and p<0.05 was considered as significant.

Total phenolic content
Total phenol content based on Gallic acid equivalency (GAE) was 52.9 ± 2.3 mg GAE/g.

Effects of T. terrestris extract on the serum levels of amylase and lipase
Administration of cerulein (50 µg/kg, i.p.) caused a surge in amylase and lipase levels in the control group in comparison to sham group (p<0.001). As it is shown in Figures 1 and 2, administration of hydroalcoholic extract of T. terrestris 400 mg/kg (i.p.) caused a significant decline in serum level of amylase and lipase (p<0.01). Besides, oral administration of T. terrestris extract 200 and 400 mg/kg markedly decreased both amylase and lipase activity (p< 0.001) (Figures 3 and  4).

Effects of T. terrestris extract on the MPO activity
As it is shown in Figures 5 and 6, pancreatic MPO activity was increased in control group (p<0.001) following induction of pancreatitis. Pre-treatment with T. terrestris extract 400 mg/kg produced a significant reduction in MPO levels in both i.p. and p.o. treated groups (p<0.001). Besides, treatment of mice with T. terrestris extract 200 mg/kg, attenuated MPO activity level just in orally treated mice ( Figure 6).

Effects T. terrestris on the histological parameters
As shown in Figure 7a, sham group exhibited a normal architecture with intact epithelium in pancreatic tissue. On the other hand, the pancreas was grossly swollen and enlarged with a visible collection of edematous fluid in pancreatitis control tissue (Figures 7b and  c). Upon microscopic examination, edema, hemorrhage, focal acinar necrosis, conspicuous vacuo1ization, and PMN infiltration in the pancreas were observed in the cerulean-treated control group (Figure 7b and c). In groups treated with T.terrestris extracts (200 and 400mg/kg) improvement in histopathology markers are detectable at different degrees ( Figure  7d, e and f).
As shown in table 1, treatment with T.terrestris extract (200 mg/kg, i.p.) attenuated leukocyte infiltration in injurious pancreatic tissue compared to control group (p<0.05). Edema and leukocyte infiltration, two inflammatory indices were also decreased following administration of T. terrestris extract 400 mg/kg (p<0.05 and p<0.01, respectively) in pancreatic injurious mice.
In groups treated with T. terrestris extract by oral route, administration of extract 200 mg/kg decreased leukocyte infiltration index in inflamed pancreatic tissue compared to control (p<0.05). Also, treatment of animals with 400 mg/kg of the extract significantly decreased edema (p<0.05) and leucocyte infiltration (p<0.01) index in treated groups (Table 2).
There was no significant change in vacuolization among the groups (Tables 1  and 2).

Discussion
The results of the present study clearly demonstrated that administration of T. terrestris hydro-alcoholic extract at doses of 200 and 400 mg/kg (p.o.) and at the dose of 400 mg/kg (i.p.) attenuated the severity of cerulein-induced pancreatitis in mice. This beneficial effect was indicated by biochemical, immunological, and histological evaluations.
Our findings showed that T. terrestris decreased MPO activity, and serum amylase and lipase levels, and improved histological parameters. Moreover, this effect was likely dependent on the dose and route of administration. It is assumed that the dose of hydro-alcoholic extract is important for its effectiveness. It means higher doses possess more effective protection activity against AP while the lower doses have negligible or insignificant effect.
The other influential parameter is the route of administration as it is manifested by the results, orally-treated mice had better response to treatment and it could be a consequence of locally active herb's components which are probably nonabsorbable through the gastrointestinal (GI) tract.
Cerulein, is an analog of cholecystokinin (CCK) which acts as an agonist for CCK1 and CCK2 receptors, so, it activates Jak/Stat pathway and generates free oxygen radicals by inducing oxidant sensitive transcription factor (Hamilton et al., 1984). In ceruleininduced pancreatitis, reactive oxygen species are produced, resulting in accumulation of destructive oxygen and conducting leukocyte and prostaglandin synthesis; So, it is reasonable that any active compound which is able to scavenge free radicals and suppress NFκB generation, diminishes the inflammatory response, and eventually improve AP (Kim, 2008). It has been shown that aqueous extract of T. terrestris can induce cell growth arrest and apoptosis by downregulating NF-κB signaling (Kim et al., 2011); So, inhibiting NF-κB signaling pathways in the pancreatic tissue could be one of the mechanisms by which this extract alleviated inflammation in AP.
The major chemical ingredients of T. terrestris are biologically active phenolic compounds like furostanol and spirostanol saponins which can trap free radicals and exhibit anti-oxidant activity (Zheleva-Dimitrova et al., 2012).
Several studies have recognized a relationship between antioxidant activity and the amount of polyphenolic compounds within the herbs (Katalinic et al., 2006;Shan et al., 2005;Zheng and Wang, 2001). In this study, total phenolic content determination of extract showed that the amount of total phenols within the T. terrestris was significantly high that can confirm this correlation. It is probable that these compounds can protect pancreatic tissue against free oxydoradicals produced by cerulein.
Antibacterial effect of T. terrestris has been reported by Kostova and Dinchev (2005). We know that AP could be associated with systemic acute bacterial infections (Beger et al., 1997), so this property of T. terrestris could support its application in a clinical setting. Antiinfection effect of T. terrestris has been attributed to DNA gyrase inhibiting property of its flavonoid contents and/or detergent-like saponins found in high amount within the extract (Bedir et al., 2002;Cushnie and Lamb, 2005;Lakhanpal and Rai, 2007). Khalid and his coworkers demonstrated that many phyto-constituents with anti-inflammatory effect were present in T. terrestris and the existence of 25 compounds was disclosed. A wide range of phytochemicals was responsible for anti-inflammatory activity of phenolic components (i.e. quercetin) and terpenoids (i.e. mono terpene lactone (−)-loliolide) that are tracked in this herb. Several mechanisms of action such as: (a) Antioxidative and radical scavenging activities (Ojha et al., 2006); (b) modulation of cellular activities of inflammation-related cells (mast cells, macrophages, lymphocytes, and neutrophils) (Mishra et al., 2013); (c) modulation of pro-inflammatory enzyme activities such as phospholipase A2 (PLA2), cyclooxygenase (COX), and lipoxygenase (LOX) and the nitric oxide (NO) producing enzyme, nitric oxide synthase (NOS) (Hong et al., 2002), have been proposed to explain the antiinflammatory actions of these phytoconstituents.
Because AP is one of the most prominent inflammatory diseases of the GI tract (Beger et al., 1997), it could be suggested that these mentioned mechanisms can modulate the severity of the disease.
On the other hand, T. terrestris extract has a high capacity to suppress experimental colitis as indicated by the macroscopic, microscopic and biochemical evaluations carried out by Rajesh et al., (2013). The authors elucidated anti-IBD (inflammatory bowel disease) properties of T. terrestris in an animal model of colitis induced by dextran sulfate sodium (DSS). It seems that the inhibition of neutrophil infiltration into the colonic mucosa by T. terrestris suppressed the inflammatory responses, which leads to the development of DSS-induced colitis (Rajesh et al., 2013); since the pathologic mechanisms underlying AP and IBD are basically similar (Rasmussen et al., 1999), anticolitis property of T. terrestris could explain its beneficial effect in AP.
In addition, it has been shown that T. terrestris has some more beneficial effects on serum lipid profile which is regularly deteriorated in AP. It reduced TG and LDL levels in experimental diet-induced hyperlipidemia (Chu et al., 2003).
Another beneficial action that has been elucidated for T. terrestris, is its analgesic effect. This effect that has been reported by Heidari et al. (2007) could alleviate the pain that is regularly associated with AP while this natural compound lacks ulcerogenic property of common NSAIDS and glucocorticoids (Graham, 2000;Heidari et al., 2007) in GI tract.
In conclusion, the hydro-alcoholic extract of T. terrestris used in this study was potent enough to protect against an experimental model of pancreatitis induced by cerulein in mice. This property, could be attributed to many active ingredients that are present in this miraculous fruit.
Several bioactivity and biological mechanisms of actions have been investigated and demonstrated for them; however, more mechanistic experiments are needed to identify the exact mechanisms that are involved.